Stimulation of mast cells via the high affinity receptor for IgE (FcepsilonRI) leads to the release of inflammatory mediators that are responsible for allergic inflammatory diseases, such as asthma and allergic dermatitis. The release of inflammatory mediators is dependent on the increase in cytosolic free Ca2+ ([Ca2+]i), which is initiated by the mobilization of Ca2+ from the endoplasmic reticulum and followed by influx of Ca2+ from outside of the cell. It is generally believed that inositol 1,4,5-trisphosphate (IP3) is responsible for mobilizing Ca2+ from the intracellular stores. However, we have shown that FcepsilonRI-mediated production of IP3 is not sufficient to account for the increase in [Ca2+]i. This suggests an alternative mechanism for Ca2+ mobilization via sphingosine 1- phosphate, a product of sphingosine kinase. Furthermore, the increase in sphingosine 1-phosphate production and Ca2+ mobilization has been observed in an antigen-stimulated mast cell line, RBL-2H3 cells that has been overexpressed with the beta isoform of Class II phosphoinositide 3-kinase (PI3KC2beta). Our long-range goal is to understand the mechanisms underlying [Ca2+]i increase in mast cells. The objective of this particular application is to understand the role of Class II PI3K in sphingosine kinase activation and Ca2+ mobilization. The central hypothesis for the proposed research is that activation of a Class II PI3K via FcepsilonRI increases production of sphingosine 1-phosphate, which causes Ca2+ mobilization in conjunction with IP3 in RBL-2H3 Cells. We formulated this hypothesis based on strong preliminary findings, which demonstrate that overexpression of PI3KC2beta increases FcepsilonRI-mediated production of sphingosine 1-phosphate and Ca2+ mobilization. In addition, increased production of sphingosine 1-phosphate leads to increased mobilization of Ca2+ only in the presence of IP3. The rationale for the proposed research is that, once the alternative pathway of Ca2+ mobilization is elucidated, it can be targeted as a new approach to treat allergic inflammatory diseases. The central hypothesis will be tested by pursuing three specific aims: 1) Identify initiating biochemical events for FcepsilonRI-mediated Ca2+ mobilization. 2) Identify intermediate signaling molecule(s) in the alternative pathway for FcepsilonRI-mediated Ca2+ mobilization. 3) Determine how FcepsilonRI-mediated Ca2+ mobilization is regulated by sphingosine kinase and phospholipase C pathways in RBL-2H3 cells. It is our expectation that the resultant approach will delineate an alternative Ca2+ signaling pathway that involves the activation of PI3KC2beta, sphingosine kinase and phospholipase C. The research proposed in this application is significant, because the outcomes are expected to provide new targets for therapeutic interventions for the prevention and treatment of allergic inflammatory diseases. In addition, they are expected to offer a better fundamental understanding of how Ca2+ is mobilized in mast cells.